Project Summary/Abstract Type 1 diabetes (T1D) is a complex autoimmune disease. Recent clinical studies show that pancreatic beta-cell dysfunction (e.g., a decline of first phase insulin release) occurs several years before the clinical onset of T1D. Unfortunately, we have little knowledge of mechanisms underlying early beta-cell dysfunction despite the essentiality of such understanding for identifying novel therapeutic targets that could potentially arrest disease development. The long-term goal of this project is to gain a detailed mechanistic understanding of early beta-cell dysfunction in T1D through advanced in situ molecular characterization of clinical tissue specimens and functional studies. Our research plan exploits the substantial intra-donor islet heterogeneity observed in both pre-diabetic and recent-onset T1D subjects. Our hypothesis is that such intra-donor islet heterogeneity reflects multiple stages of progressive ?-cell dysfunction, with each islet responding as a cohort of cells to its unique microenvironment. Identifying the molecular signatures of each stage presents a novel opportunity to elucidate mechanisms of early ?-cell dysfunction in T1D. To identify the molecular determinants of each stage of early ?- cell inflammation and stress, proteomics characterization is considered as critical because the expressed proteome is directly connected to the phenotype. We will first conduct ultrasensitive proteomics analysis of single islets isolated by laser microdissection from multiple autoantibody positive (AAb+) donors. We will also conduct deep proteomics profiling of pooled islet subpopulations (e.g., 10-20 islet sections per pool) expressing defined immunohistochemical (IHC) markers to identify crucial regulatory pathways of early ?-cell dysfunction. Our approach is enabled by the recently developed innovative nanoPOTS (Nanodroplet Processing in One-pot for Trace Samples) technology for single islet proteomics and deep proteome profiling. Specifically, in Aim 1 we will pursue single islet proteomics profiling of presymptomatic multiple AAb+ donors to identify molecular signatures of progressive ?-cell dysfunction in the metabolic, secretory, and stress pathways. In Aim 2, we will focus on determining which stress and inflammatory pathways are most tightly associated with early ?-cell inflammation through deep proteome profiling comparing islet subpopulations defined by known IHC markers. Aim 3 will explore the functional significance of identified regulators through assessments of their roles in cell survival, apoptosis and secretory function. The overall proteomics data will be integrated with transcriptomics and other experimental data to identify candidate regulators for functional studies. Statement of Impact: We anticipate this project will advance the field by establishing first-of-its-kind molecular resource on the mechanisms of islet heterogeneity and early ?-cell dysfunction in the progression to T1D and demonstrate an effective path towards rational selection of therapeutic targets that can drive future preventive strategies.